DESCRIPTION: Enzymes achieve large accelerations in the rate of a chemical reaction. The investigator's long-term goal is to understand the sequence of reaction steps that define an enzyme mechanism, as well as the interactions in the enzyme active site that lead to those observed rates. The system chosen for study in this application is (S)-mandelate dehydrogenase (MDH), an enzyme from Pseudomonas putida. MDH is a member of a large family of FMN-dependent hydroxy acid oxidases/dehydrogenases ranging from mammals to bacteria. Flavin-dependent enzymes which oxidize a hydroxy or an amino acid to the corresponding keto acid are ubiquitous. They share highly similar reaction mechanisms which involve carbanion intermediate formation followed by electron transfer to the flavin. Even though carbanions are mechanistically difficult to generate, they occur commonly in both flavin and non-flavin enzymes. The immediate objective of this application is to understand the mechanism by which carbanion intermediates are generated and stabilized in flavoproteins, and the mechanism by which electrons are transferred from these intermediates to the flavin cofactor.